江汉油田区典型农田土壤烃类降解微生物功能基因bssA的遗传多样性研究

摘要

油田区土壤易受烃类物质影响并可能富集了特异的石油烃降解微生物类群.针对江汉油田区5个不同油井口附近的典型旱地农田土壤,采用石油烃(Petroleum hydrocarbons,PHs)中苯系物代谢的关键功能基因-bssA(苯甲基琥珀酸合成酶基因)作为分子标识物,通过克隆文库结合末端限制性片段长度多样性(Terminal-restriction fragment length polymorphism,T-RFLP)的方法,研究该油田区土壤含有bssA基因的烃类降解微生物群落结构,并探讨其环境驱动机制.结果表明,土壤中PAHs含量在0.21~2.01 mg kg-1之间,石油烃污染程度较低.T-RFLP的分析表明不同土壤样品中的bssA基因多样性差异明显,PAHs(Polycyclic Aromatic Hydrocarbons,多环芳烃)含量最高土壤中bssA基因多样性最高,其优势bssA基因类群与硫酸盐还原菌或地杆菌有较近的亲缘关系.冗余分析进一步表明,土壤硝态氮、有效磷、PAHs含量均是影响bssA基因多样性的重要因子.这些结果表明:江汉油田区典型农田土壤中含有bssA基因的主要类群为β-变形菌和δ-变形菌,并与地杆菌属(Geobacter)、索氏菌属(Thauera)和固氮菌属(Azoarcus)具有较近的系统发育亲缘关系.这些微生物可能通过硝酸盐、硫酸盐及铁还原代谢过程降解土壤PAHs.%Farmland soils in oil fields are liable to get polluted with petroleum hydrocarbons(PHs), which consequently leads to enrichment of some special PHs degrading microbial groups in the soils. In this study,soil samples were collected from typical farmland fields near five different oil wells in the Jianghan Oilfield located in Qianjiang City,Hubei Province for analysis of PHs degrading microbial groups,using bssA (benzylsuccinate synthase gene),a functional gene key to anaerobic benzenes degradation as biomarker, and community structure of the PHs degrading microbial groups was determined with T-RFLP(Terminal-restriction fragment length polymorphism)and clone library. On such a basis,environmental factors affecting community composition of the soil bssA were discussed. Results show that the content of polycyclic aromatic hydrocarbons(PAHs)in the soil varied in the range of 0.21~2.01 mg kg-1,which indicates that the soil in this region was not seriously contaminated with PHs. Meanwhile,the T-RFLP analysis indicates that bssA diversity varied significantly from soil sample to soil sample in this oilfield,being the highest in the soil sample the highest in PAHs content,and its dominant group was in a fairly close kinship with sulfate-reducing bacterium or Geobacter. Furthermore,RDA(Redundancy analysis)reveals that the contents of soil available nitrogen,phosphorus and PAHs were all main factors affecting soil bssA diversity. To sum up,all the findings indicate that the bssA-bearing microbial groups in the soil of this oilfield are Beta-proteobacteria and Deta-proteobacteria,and in close kinships with Thauera,Azoarcus and Geobacter in phylogenesis. All these microbes might degrade PAHs through reducing metabolic processes of nitrate,sulfate,and iron.